Fall 08 TargetingT argeting Epidermal Growth Factor to treat pregnancy complications Roxanne Hastie BioMedSc, BSc (Hons) Department of Obstetrics and Gynaecology Faculty of Medicine, Nursing and Health Science University of Melbourne February 2018 Submitted in total fulfilment of the requirements of the degree of Doctor of Philosophy ORCID identifier: 0000-0003-3458-6831 Abstract Epidermal growth factor receptor (EGFR) signaling is one of the most avidly studied signaling networks in mammalian biology, with critical roles in cellular growth and survival. The EGFR is also vital for normal placental formation and development. This body of work examines the role and potential of targeting the EGFR cascade in the pregnancy complications preeclampsia and ectopic pregnancy. In the first arm of this PhD, we investigated EGFR signaling in preeclampsia, a major pregnancy complication associated with shallow placental invasion and placental insufficiency. Anti-angiogenic factors are then released from the preeclamptic placenta into the maternal circulation causing endothelial dysfunction (a hallmark of the disease), hypertension and organ injury. Firstly, we examined whether administering EGF peptide (the natural EGFR ligand) to primary endothelial cells affects endothelial dysfunction in-vitro. From this, we found EGF peptide to significantly reduce a number of markers of endothelial dysfunction in-vitro, potentially suggesting this natural ligand might offer a therapeutic approach to treat the endothelial dysfunction characteristic of preeclampsia. Over the last two decades the understanding of this disease has increased greatly with the discovery of the anti-angiogenic molecules soluble Fms-like tyrosine kinase-1 (sFLT-1) and soluble endoglin (sEng). Theses factors are released at excessive levels from the preeclamptic placenta and cause the widespread maternal endothelial dysfunction that gives rise to the multi-organ injury that occurs in preeclampsia. However, upstream mechanisms regulating 2 the release of these molecules, while avidly sought, are still poorly described. In this thesis, we describe a series of studies that demonstrate EGFR is overactive in preeclampsia, and is a positive upstream regulator of sFLT-1 and sEng. We first characterized EGFR signaling in a large cohort of severe preterm preeclamptic placentas, compared to gestation matched normotensive controls. We found not only EGFR signaling to be significantly increased in preeclamptic placenta, but also confirm that there is increased signaling of the key downstream EGFR adaptor molecules: ERK1/2, Akt and STAT-3. We then proceeded to functional experiments in primary trophoblast (placental) cells to investigate whether manipulating EGFR (and its downstream molecules) affected the release of sFLT1 and sEng. Indeed, we found activating EGFR signaling in placenta increased sFLT-1 secretion and conversely, inhibiting multiple points of the EGFR signaling cascade using either small molecule inhibitors or siRNAs significantly and dose-dependently reduced primary trophoblast sFLT-1 and sEng secretion. We progressed with an in-vivo experiment and confirmed that administration of gefitinib, a small molecule EGFR inhibitor, to pregnant mice significantly reduced circulating levels of sFLT-1. We previously identified sulfasalazine, esomeprazole and statins decrease sFLT-1 secretion, but were yet to uncover the molecular mechanism by which these drugs were affecting sFLT-1 secretion. We therefore investigated EGFR signaling in primary trophoblasts after treating with sulfasalazine, esomeprazole and statins, and found these drugs that reduce sFLT-1 secretion also significantly down regulate EGFR and downstream adaptor molecule expression. Together, this body of work suggests EGFR signaling positively regulates sFLT-1 secretion. The clinical implication is that blocking placental EGFR signaling may be a novel therapeutic 3 approach to treat preeclampsia. The second arm of this thesis was to investigate therapeutics for ectopic pregnancy, and is relevant to the theme of this thesis given EGFR inhibitors have become a leading therapeutic candidate to treat this condition. Ectopic pregnancies arise from implantation of an embryo outside of the uterus, most commonly in the Fallopian tubes. They represent a medical emergency as they can rupture, causing fatal internal bleeding. The majority of ectopic pregnancies are surgically excised, in part owing to the inefficacy of the only available medical treatment, methotrexate. Previous work has shown combining methotrexate with the EGFR inhibitor, gefitinib to be additive at resolving ectopic pregnancy, however this combination is yet to prove successful for large ectopic pregnancies, which still require surgery. Therefore, in an attempt to improve the medical management of ectopic pregnancy, within the second arm of this study we investigated therapeutics, either as single agents or in combination with gefitinib (an EGFR inhibitor) that could provide superior efficacy to methotrexate or combination methotrexate and gefitinib to treat ectopic pregnancy. We screened a number of chemotherapeutics for their ability to induce placental death in-vitro, beyond that of methotrexate and the combination of methotrexate and gefitinib (which is currently been assessed in a stage III clinical trial). From this screening we identified vinorelbine, a well tolerated chemotherapeutic, to be 100 to 1000 times more potent than either methotrexate or combination methotrexate and gefitinib at inducing placental cell death. Importantly, vinorelbine is a tablet, which makes it a highly attractive drug option to treat ectopic pregnancy. Thus, we further investigated vinorelbine in-vivo using a mouse placental xenograft model and found vinorelbine to be significantly more efficacious at 4 resolving placental xenografts than either methotrexate or combination methotrexate and gefitinib. Importantly, vinorelbine did not impact upon subsequent fertility in mice. Lastly, we assessed the potential of combining low dose vinorelbine with the EGFR inhibitor, gefitinib. This combination proved additive, causing placental cell death beyond either drug alone. In conclusion, this thesis has explored the role of EGFR signalling and potential of targeting this cascade in preeclampsia and ectopic pregnancy. Notably, we describe EGFR signalling to positively regulate placental sFLT-1 secretion and may therefore present as a novel molecular target to identify therapeutics for preeclampsia. Furthermore, we have uncovered vinorelbine, an orally available and well tolerated therapeutic, for ectopic pregnancy that has potential to be clinically translated and be a more effective treatment of ectopic pregnancy (either alone, or in combination with the EGFR inhibitor gefitinib). It is my hope that this body of work will further the field of obstetrics and gynaecology and lead to improved maternal outcomes. 5 Declaration This is to certify that: i. The thesis comprises only my original work towards the PhD except where indicated in the preface, ii. Due acknowledgment has been made in the text to all other materials used; and iii. The thesis is fewer than 100,000 words in length, exclusive of tables, maps, bibliographies and appendices. Mrs Roxanne Hastie Rhastie PhD Candidate 6 Preface Pursuant to the regulations governing the degree of Doctor of Philosophy at the University of Melbourne, I hereby submit that: I. This thesis contains no material that has been accepted for the award of any other degree or diploma in any university. II. To the best of my knowledge and belief, this thesis contains no material previously published or written by another person, except where due reference has been made. Chapters 3 and 8 of this thesis contain findings that were published in peer- reviewed journals and result from the collaboration with others in the scientific community. I declare that I am the primary author of these publications and have contributed > 50% of the work presented. The following table outlines the specific contributions of all co-authors of these publications. In addition, declaration for thesis with publication forms for each paper have been submitted with this thesis, along with signed co-author collaborator authorisation documents confirming collaborator contributions and that my contributions were > 50%. 7 Table 1: Contributors of all co-authors to published or in-review works presented in this thesis Thesis Publication Co-author roles and chapter contribution (%) Chapter 3 Title: Epidermal Growth Roxanne Hastie – Design, Factor Rescues optimisation and performance of Endothelial Dysfunction laboratory experiments, data in Primary Human analysis, manuscript preparation Tissues In-vitro. and submission, including Journal: Reproductive addressing reviewer comments Sciences. (80%) Year: 2017 Natalie Hannan – intellectual Volume: 24 1 support (3%) Pages: 245-52 Ping Cannon – technical support (2%) Stephen Tong – Intellectual support and editing of paper (7.5%) Tu’uhevaha Kaitu’u-Lino – Intellectual and design support and editing of paper (7.5%) Chapter 8 Title: Vinorelbine Roxanne Hastie – Design, potently induces optimisation and performance of placental cell death, laboratory experiments, data 8 does not harm fertility analysis, manuscript preparation and is a potential and submission (80%) treatment for ectopic Elgene Lim – intellectual support pregnancy. (1%) Journal: EBioMedicine. Pavel Sluka – technical support Year: 2018 (3%) Lisa Campbell – technical support